1. Field
The disclosed embodiments relate to a fluorescence imaging device such as those used in fluorescence analysis of biochips. More specifically, the disclosed embodiments relate to means for ensuring the various movements necessary in the course of the analysis in order to scan the surface of a chip point by point by locating each point, during its analysis, in the focal plane of a confocal microscope.
2. Brief Description of Related Developments
The analysis of the deoxyribonucleic acid, or DNA, of living organisms is of major importance in modern biology, as much for advancing knowledge of biological mechanisms as for detecting certain illnesses associated with faults in cell function.
A widespread technique for analyzing a DNA sample consists in producing biochips that are analyzed by means of fluorescence techniques.
The production of biochips is known. Its essential steps consist in depositing samples of DNA fragments to be studied on a lamella, in mixing these samples with separately prepared targets consisting of DNA labeled with fluorochromes, i.e. molecules that emit light by fluorescence when they are themselves excited by an energy source, especially excitation light, and in hybridizing the DNA to be studied with the labeled DNA. In the course of the hybridization, some strands of labeled DNA will couple with their complementary sample of DNA to be studied and thus render the strands of the DNA studied that correspond to the labeled DNA detectable by fluorescence.
The fluorochromes may be of several types and more often than not dyes that are fluorescent in red light, the dye called Cy5, or in green light, the dye called Cy3, are used.
Generally, many samples are deposited on a biochip, which constitute an array of rows and columns at the intersection of which the samples to be analyzed are found. A sample represents a “patch” or “spot” of around 50 micrometers in diameter and on a biochip carried by a traditional microscope lamella, typically 26 mm wide by 75 mm long, around 30 000 spots are frequently found.
To analyze such biochips use is made of reading devices that illuminate the samples of the biochip point by point and record the fluorescence response of each point to provide a fluorescence map of the biochip. To detect the fluorescence response at the scale of the sample DNA strand, it is necessary for the reading device to have a resolution of around 10 micrometers or less.
There are such reading devices that employ a microscope called a “confocal” microscope and a scanning system that enables the surface of the biochip to be read in a reasonable time while analyzing this surface point by point.
The confocal microscope is an optical microscope often coupled with fluorescence techniques that uses monochromatic light (most often coming from a laser source) to illuminate the sample and observe the response of said sample through a pinhole, which has the effect of practically limiting the area of the sample observed to the focal plane of the objective, often less than 2 micrometers deep, and to confer on the microscope a high resolution close to the theoretical maximum for a microscope using photons in the visible region. To compensate for the loss in luminosity due to the small opening of the pinhole, observation is carried out by means of a photomultiplier, the measurements of which are recorded in order to reconstruct the images of the sample observed point by point by conventional imaging means, for example on a computer.
The very low depth of field intrinsic to the confocal microscope makes it necessary, during its use to read chips, to control the position of the focal point of the microscope in order that the area illuminated by the excitation laser and its fluorescent response be correctly positioned despite irregularities in the surface of the chip and the uncertainties in the position of this surface.
To control the position of the focal point, the known imaging devices use active control means to control the lens or the group of lenses of the microscope objective. This lens or group of lenses is mounted in a movable manner and its position is permanently adjusted along the optical axis of the objective by an actuator such as a magnetic coil or a piezoelectric actuator in response to a focusing error signal. This movable assembly is particularly fragile and sensitive to impacts and is likely to be disturbed.
To read all the points of the chip successively and reconstruct a fluorescence image of each of the samples to be analyzed, the chip is subjected to scanning by the focal point of the confocal microscope.
Conventionally, the surface of the chip is scanned along its transverse axis by means of rapid displacement of the objective of the confocal microscope and the chip support moves at a lower speed along a perpendicular axis, the longitudinal axis, so that the objective describes a series of approximately parallel reading lines, until the entire surface of the chip has been scanned. In some models of biochip analyzer, the objective of the microscope is carried by a carriage guided on rails along a straight path, moved by a linear motor as described for example in the U.S. Pat. No. 5,459,325 or moved by a rotating motor by means of a connecting rod assembly as shown by the figures of the Patent JP 2003 185583. In other models, such as for example described in the U.S. Pat. No. 6,201,639, the microscope objective is carried by an arm undergoes an oscillating rotational movement about a vertical axis and describes a path in a circular arc above the biochip.
This type of mounting of the microscope objective proves to be relatively expensive to produce due to the cost of the components themselves, such as that of the linear motors, or due to the complexity of the movements in order to obtain a constant scanning rate. The complexity of these elements is also increased when the means of focusing the confocal microscope objective are mounted on the movable part, which increases the mass thereof.
Furthermore, the delicate focusing means linked to the microscope objective must take account of the fact that the objective is movable and is subject to frequent accelerations each time the scanning changes direction.